Molecular basis for the preferential cleft recognitionby dromedary heavy-chain antibodiesErwin De Genst*†, Karen Silence*‡, Klaas Decanniere*, Katja Conrath*, Remy Loris*, Jorg Kinne§, Serge Muyldermans*,and Lode Wyns*

*Department of Cellular and Molecular Interactions, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Pleinlaan 2,B-1050 Brussels, Belgium; §Central Veterinary Research Laboratory, P.O. Box 597, Dubai, United Arab Emirates; and ‡Ablynx, Technologiepark 4,B-9052 Zwijnaarde, Belgium

Edited by Martin G. Weigert, University of Chicago, Chicago, IL, and approved January 24, 2006 (received for review June 28, 2005)

Clefts on protein surfaces are avoided by antigen-combining sitesof conventional antibodies, in contrast to heavy-chain antibodies(HCAbs) of camelids that seem to be attracted by enzymes’ sub-strate pockets. The explanation for this pronounced preference ofHCAbs was investigated. Eight single domain antigen-bindingfragments of HCAbs (VHH) with nanomolar affinities for lysozymewere isolated from three immunized dromedaries. Six of eightVHHs compete with small lysozyme inhibitors. This ratio of activesite binders is also found within the VHH pool derived frompolyclonal HCAbs purified from the serum of the immunizeddromedary. The crystal structures of six VHHs in complex withlysozyme and their interaction surfaces were compared to those ofconventional antibodies with the same antigen. The interface sizesof VHH and conventional antibodies to lysozyme are very similaras well as the number and chemical nature of the contacts. Themain difference comes from the compact prolate shape of VHH thatpresents a large convex paratope, predominantly formed by the H3loop and interacting, although with different structures, into theconcave lysozyme substrate-binding pocket. Therefore, a singledomain antigen-combining site has a clear structural advantageover a conventional dimeric format for targeting clefts on antigenicsurfaces.

antibody–lysozyme structures � camel single domain antibody � enzymeinhibitor � epitope–paratope interactions

S ix hypervariable antigen-binding loops constitute the anti-gen-combining sites of conventional antibodies. These loops,

three (H1–H3) from the variable domain of the heavy chain(VH), three (L1–L3) from the variable domain of the light chain(VL) are juxtaposed forming a continuous surface (paratope)that is complementary to a surface on the antigen (epitope) (1).The paratope is essentially planar for protein antigens and formsa groove or cavity to interact with peptides and haptens (2, 3).The loops L1–L3 and H1–H2 fold into a limited number ofcanonical structure classes, determined by the loop length andthe presence of conserved residues at key positions within thehypervariable and framework regions (4, 5). The extreme lengthand sequence variability of H3 makes the structure prediction ofthis loop extremely difficult (6).

The structures of antigen-binding sites and loops, as well as thecanonical loop determining residues, are well established (1, 4,5). In contrast, the elucidation of the molecular basis for therecognition of particular epitopes by antibodies remains a majorchallenge. Our knowledge and paradigms of protein–epitoperecognition by antibodies is largely based on the analysis of theimmune response toward hen egg white lysozyme (HEWL). Thisis due to the high antigenicity, the large number of naturalvariants of HEWL (7), and the availability of eleven differentcrystal structures of Fab or Fv antibody fragments (8–10) incomplex with lysozyme, collected over the last two decades. Sixstructures represent Fabs or Fvs that are clearly clonally unre-lated (8), and one additional structure involves an artificiallyassembled antigen-specific VH–VL pair (11).

Camelids possess a functional class of antibodies devoid oflight chains (referred to as heavy-chain antibodies or HCAbs)(12, 13). The antigen-combining site of these heavy-chain anti-bodies is limited to only three hypervariable loops (H1–H3)provided by the N-terminal variable domain (VHH). The firstcrystal structures of VHHs revealed that the H1 and H2 loopsare not restricted to the known canonical structure classesdefined for conventional antibodies (14). The H3 loops of VHHsare on average longer than those of conventional antibodies (15).In one case, the H3 loop was shown to protrude from theremaining paratope and inserts into the active site cleft ofHEWL (16–18). Furthermore, it seems that a large fraction ofthe dromedary HCAbs have a preference for binding into activesites of enzymes against which they were raised (19).

To elucidate the structural basis of the remarkable HCAbepitope preferences, we performed an in depth analysis ofepitope recognition by HCAbs in analogy to the study done forconventional antibodies toward HEWL (7, 8). We immunizedthree different dromedaries with HEWL, isolated eight anti-HEWL VHHs via phage-display, and mapped their epitopes.These epitopes of the isolated VHHs cluster in two nonover-lapping regions with the vast majority (six of eight) binding intothe enzyme’s active site cleft. The same proportion (�85%) ofactive site binders was also observed within the polyclonal HCAbimmune response toward HEWL. The crystal structures of sixVHHs in complex with HEWL demonstrate that the paratopesare characterized by a variety of different structures, dominatedby the H3 antigen-binding loop. Our results sharply contrast withthe epitope preferences of murine anti-HEWL responses rep-resented by the available Fab or Fv::lysozyme crystal structures(8), which display planar epitopes located at three patchesoutside the active site.

ResultsOntogeny of the Isolated VHHs. Eight different anti-HEWL VHHs,originating from three different dromedaries were isolated fromimmune libraries. The hypervariable loops of these VHHs differsignificantly in sequence (Fig. 5, which is published as supportinginformation on the PNAS web site). Especially the sequencesand lengths (12–24 aa) of the H3 region vary considerably.VHHs, like VH domains, are assembled through the recombi-nation of one variable (V), one diversity (D), and one joining (J)gene segment out of a pool that is present in the genome (20).A search in the VHH germ-line gene database (20) revealed that

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: VH, heavy-chain variable domain; VL, light-chain variable domain; HEWL,hen egg white lysozyme; HCAb, heavy-chain antibody; VHH, variable domain of a heavy-chain antibody.

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,www.pdb.org (PDB ID codes 1ZVY, 1ZVH, and 1ZV5).

†To whom correspondence should be addressed. E-mail: edegenst@vub.ac.be.

© 2006 by The National Academy of Sciences of the USA

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the V gene segments of these HEWL-binders most probablyoriginated from either the cvhhp08 (AJ245114.1) or the cvhhp11(AJ245117.1) germ-line genes. These V-genes are the mostfrequently used germ-line genes in the V-D-J recombination ofVHHs (20). Nucleotide sequence identities between maturedand germ-line V sequences range from 84.4% (D2-L31) to 94.1%(D2-L27), corresponding to 17–39 nucleotide substitutions (Fig.5). These substitutions follow the characteristic pattern for aclassical somatic hypermutation mechanism leading to antigenaffinity maturation. The analysis further indicated that the Jsegments JH3 or JH5 were used in the V-D-J rearrangement toarrive at the VHHs (camelid JH germ-line sequences; V. K.Nguyen, personal communication).

Kinetics and Affinity of the VHH–HEWL Interactions. The kinetic andaffinity constants of all VHH–HEWL interactions were deter-mined by surface plasmon resonance (Fig. 6, which is publishedas supporting information on the PNAS web site). For the VHHsD3-L11, cAb-Lys-3, cAb-Lys-2, D2-L19, D2-L29, and D2-L24,the ka constants are within a range of 1 order of magnitude(0.21–5.4 � 106 M�1�s�1) and the kd values cover a range of twoorders of magnitude (1.7–200 � 10�4 s�1). The affinity constants(KD) range from 77 pM to 70 nM. The kinetics of the D2-L27-HEWL interaction was too fast to measure. Nonlinear regressionof the equilibrium binding response versus D2-L27 concentra-tion yielded an affinity constant of 2.34 �M. The D2-L31–HEWL interaction exhibited complex binding kinetics consistentwith an induced fit mechanism, with a global affinity constant of69 nM. The ka, kd, and KD values for these VHH–HEWLinteractions are within the range found for matured conventionalantibody–antigen interactions (21).

Anti-HEWL VHHs Bind Predominantly to the Active Site Cleft of HEWL.Using surface plasmon resonance (see Methods) we mapped theregions on the surface of HEWL that are recognized by ourVHHs. The VHHs D2-L19, D2-L29, D2-L31, D3-L11, cAb-Lys-2, and cAb-Lys-3 (group 1) mutually exclude each other forantigen binding (Fig. 1A and Table 1, which is published assupporting information on the PNAS web site). The VHHs,D2-L24 and D2-L27 (group 2) also interfere with each other forantigen binding but remain competent to associate with HEWLin the presence of the VHHs of group 1 (Fig. 1 A and Table 1).The small chemical compounds GlcNAc(�1–4)GlcNAc(�1–4)GlcNAc (NAG3) occupying the A-C substrate subsites ofHEWL and Biebrich Scarlet, associating with the D-F carbohy-drate-binding sites (22), inhibit the cAb-Lys-3, D2-L31, andD3-L11 interaction with HEWL. Only Biebrich Scarlet affectsthe HEWL binding of D2-L19, cAb-Lys-2, and D2-L29 (Fig. 1B

and Table 1). Therefore, the assembly of all epitopes of group 1VHHs covers a large part of the substrate-binding site oflysozyme.

The dromedary HCAb isotypes, IgG2 and IgG3, are easilyseparated from conventional antibodies, IgG1 (12). A proteo-lytic digestion of these IgG3 antibodies (the most abundantHCAb isotype in the dromedary) allows the purification of apolyclonal VHH fraction (19). Such VHH pool of a dromedaryimmunized with HEWL was competed with D2-L19 (epitopegroup 1 member) or D2-L24 (group 2 member) for HEWLbinding. Fig. 1C shows the percentage of binding in the absence(100%) or presence of monoclonal competitor, as a mean ofthree experiments at different concentrations of polyclonalserum VHHs. The group 2 and 1 VHHs inhibit the HEWLbinding of the polyclonal VHH pool by �25% and �85%,respectively. Therefore, all HEWL-binding VHHs of IgG3 ofdromedary D2 belong to one of the two observed complemen-tation groups with the majority belonging to group 1. Theseepitope mapping experiments clearly show that the HCAbimmune response is directed predominantly toward the activesite of HEWL and that our set of monoclonal anti-HEWL VHHscontains a similar proportion of active cleft binders.

The VHH::HEWL Complex Structures. To allocate the differentepitopes on HEWL at atomic resolution and to clarify thestructural details of the paratope–epitope associations, we crys-tallized six VHH::HEWL complexes (Fig. 2). The structureswere solved to a resolution varying between 1.4 and 2.1 Å andrefined to Rfree factors between 0.196 and 0.239 (Table 2, whichis published as supporting information on the PNAS web site).

All VHHs possess the typical V Ig fold. The H1 loops adoptconformations that deviate significantly from the conventionalcanonical structure (Fig. 7A, which is published as supportinginformation on the PNAS web site), whereas the H2 loops adoptclassical canonical structures (Fig. 7B). The H3 loops of allVHHs fold onto the five-stranded �-sheet and interact withresidues of the framework 2 regions (Fig. 7C). This structuralmotif is a typical feature found in VHHs. It stabilizes the long H3loops (23) and blocks access to the side of the domain corre-sponding to the VL interaction site of a conventional VH domain(13, 17).

The VHHs D2-L19, cAb-Lys-2, and D2-L29 contact highlyoverlapping epitopes (Fig. 2 Right), located at the D-F carbo-hydrate substrate subsites of HEWL, including the HEWLcatalytic residues Glu-35L and Asp 52L (Fig. 2 A–C and Table 3,which is published as supporting information on the PNAS website). D2-L29 buries the side chain of Thr-47L (located at thelower edge of the catalytic site) deeply between the H3 loop and

Fig. 1. Epitope mapping of the monoclonal and polyclonal VHHs. (A) HEWL binding of all VHHs in the presence of a saturating concentration of cAb-Lys-3 usinga coinjection procedure. (B) Epitopes of HEWL active site binders based on the inhibition of binding by NAG3 and Biebrich Scarlet. (C) Residual HEWL bindingof polyclonal VHH derived from the IgG3 fraction of dromedary D2. (Left) Binding in absence of competitor (set at 100%). (Center) Binding in presence ofHEWL-saturating concentrations of D2-L24. (Right) Binding in presence of HEWL-saturating concentrations of D2-L19.

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the framework 2 region (Fig. 2 A). This allows Arg100e of H3 toform a salt-bridge and two hydrogen bonds with Glu-35L and twowith Asp-52L. D2-L19 and cAb-Lys-2 bury Thr-47L betweentheir H3 and H2 loops (Fig. 2 B and C), which again allowresidues of the H3 loops of both binders to make intimate contactwith Glu-35L and Asp-52L (Fig. 2 B and C). The most remarkabledifference between D2-L29 (Fig. 2A) and D2-L19�cAb-Lys-2(Fig. 2 B and C) is found in the relative orientation to HEWL.In the former case, the H2 loop is more distant from the antigenand the H1 loop contacts the opposite side of the active site cleft.In the latter two cases, the HEWL is bound around the compactprolate VHH, without any involvement of the H1 loop forantigen binding.

The VHHs cAb-Lys-3 and D3-L11 target overlapping epitopeslocated at the A-D carbohydrate subsites of HEWL and includethe residues Trp-62L and Trp-63L (Fig. 2 D and E and Table 3).The VHHs possess different loop structures and bind to theirepitopes in a different orientation. The H1 and H2 loops ofcAb-Lys-3 contact the W62L side of the catalytic cleft, whereasthose of D3-L11 contact residues across the active site. Thepositioning of the antigen contacting residues results in a wedge-shaped paratope for both VHHs. Part of the H3 loop ofcAb-Lys-3 even protrudes from the remaining paratope andpenetrates more deeply into the active site cleft, allowing the sidechain of Ser100a to contact the catalytic residues Asp-52L andGlu-35L.

The structure of the D2-L24–HEWL complex confirms theexistence of an epitope, distinct from those of the active sitebinding group. This epitope is located outside of the active siteand involves the short �-helix 79L-84L.

VHH::HEWL Complexes Versus Fv::Lysozyme Complexes. The HEWLsuperposition of six VHH::HEWL complexes gives a strikinglycontrasting picture when compared to a corresponding super-position of the 6 representative Fv::lysozyme structures (Fig. 3)(8). The epitopes for the murine Fv’s cluster in three regions onthe lysozyme surface, each of which involves patches outside theactive site. The anti-HEWL VHHs cluster at two regions, one in

ribbons, with the framework region painted in yellow and the H1, H2, and H3loops painted in green, cyan, and red respectively. The most important aminoacids that contact E35, D52, W62, or W63 of HEWL are represented as sticksand are labeled (italic). (Right) The surface on HEWL within 5-Å distance of theVHH is color-coded according to the atom type: C, yellow; N, blue; O, red; S,orange.

Fig. 2. The VHH::HEWL complex structures. The D2-L29::HEWL (A),D2-L19::HEWL (B), cAb-Lys-2::HEWL (C), cAb-Lys-3::HEWL (D), D3-L11::HEWL(E), and D2-L24::HEWL (F) complexes are shown. (Left) VHH::HEWL complexesare represented with HEWL molecules as gray space-filling models. The activesite residues E35, D52, W62, and W63 are labeled and side-chain atomscolor-coded: C, yellow; N, blue; and O, red, for all panels. For A–C, the residueT47 of HEWL is additionally colored and labeled. The VHHs are represented as

Fig. 3. Superposition of the antibody–lysozyme complexes for conventionalantibodies (Left) and for VHHs (Right). The HEWL molecules (gray surfaces) areshown in the same orientation for both antibody classes. The antibody mol-ecules are represented as colored ribbons and their identity is indicated in thesame color.

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the active site representing the majority of VHHs and onelocated outside the active site, representing the smaller group ofanti-HEWL VHHs (represented by D2-L24, Fig. 3). To explainthis remarkable difference in epitope recognition, we examinedthe structural and chemical features of the VHH::HEWL andFv::HEWL interfaces. The statistical significance of the ob-served differences between the measured parameters of thedifferent antibody classes was evaluated by using a Mann–Whitney U test.

The �ASA of the VHH or Fv and lysozyme upon complex

formation, as well as the polarity of the interfaces (Tables 4 and5, which are published as supporting information on the PNASweb site) is comparable (P � 0.1). Also, the number of contacts,hydrogen-bonds and ion-pairs (Tables 4–6, which are publishedas supporting information on the PNAS web site) do not differsignificantly (P � 0.1) between VHH::HEWL and Fv::lysozymecomplexes. In contrast to the anti-HEWL Fvs, where the para-tope areas are more or less equally distributed over all sixantigen-binding loops, the VHHs use predominantly their H3loop for antigen interaction. The amino acids residing in the H3loops of the VHHs dominate to a large extent the �ASA of theVHH (�60–80% of total interacting area) (Fig. 4A). In addition,the paratopes (and epitopes) of all VHHs, except D2-L24,deviate significantly from planarity. The �ASAs of the VHHparatopes to HEWL are strongly dependent on the planarityindex of the VHH paratopes (Fig. 4B), whereas for Fvs thisdependency is less pronounced. Higher interface complemen-tarities, as measured by the Sc parameter, further differentiatethe VHH::HEWL interfaces from the Fv::lysozyme interfaces(P � 0.05) (Fig. 4C).

DiscussionEight matured antigen-specific VHHs were isolated from threeHEWL-immunized dromedaries by phage display. The VHH-HEWL kinetics and affinities are in the same range (i.e., nM) asthose observed for the conventional Fab–HEWL interactions,and one VHH was found to interact via an induced fit mecha-nism. This finding shows that the VHH–HEWL interactions areas potent and display similar recognition mechanisms comparedto Fab–lysozyme interactions.

The VHHs recognize two independent regions on HEWL.One region is targeted by six of eight VHHs and is composed oftwo overlapping epitopes encompassing a large part of thesubstrate-binding pocket of lysozyme. The two remaining VHHstarget a region located at the antipode of the active site. This setof anti-HEWL VHHs covers the epitope specificities of HCAbsin the serum of immunized dromedaries. Indeed, one monoclo-nal VHH (D2-L19) representative for the six active site binderscould inhibit �85% of the HEWL binding of polyclonal VHHsprepared from serum IgG3 of HEWL-immunized dromedaries.A binder (D2-L24) of the other epitope-group blocked �25% ofthe polyclonal VHHs binding to HEWL (Fig. 1C). Therefore, thedistribution of our eight VHHs over the two epitope groups isalso present to the same proportion in the polyclonal VHH pool.Furthermore, the variety in sequence and length of the hyper-variable loops of the eight VHHs proves that they are clonallyunrelated and their broad range in affinity and kinetics arguesagainst a biased selection during phage-display pannings.

Clearly, the largest fraction of the anti-HEWL VHHs interactswith the active site of the antigen. This HEWL epitope prefer-ence deviates strikingly with that of matured murine antibodiesagainst lysozyme (Fig. 3). The murine humoral response isdominated by three noncontinuous and nonoverlapping regions,located on the surface of lysozyme outside of the active site (8,24). However, the antibody HyHEL10 inhibits the hydrolyticactivity of cell wall substrates of Micrococcus luteus by 95% (25).This inhibition results from a close contact between side chainsat the periphery of the paratope with the catalytic cleft residuesTrp-62L and Trp-63L, whereas the central part of the antibody–antigen interface is located further away from the substratepocket. The VH domain of D11.15 binds also close to the bordersof the active site cleft, although it does not penetrate into thiscavity nor does it block the access to substrate binding (26).Hence, the mouse Fv::lysozyme paradigm fits perfectly with theproposal of conventional antibodies having paratopes that areessentially planar to interact with proteinaceous antigens (1–3,27). It should be noted that most of the antibody structures in theprotein database are murine antibodies and these antibodies,

Fig. 4. Structural and chemical differences between VHH::HEWL andFv::lysozyme interfaces. (A) Contribution of each antigen-binding loop to the�ASA of the paratope. (B) The planarity index of the VHH or Fv paratope versustheir �ASA. The dotted lines represent the linear trends for both types ofantibodies (VHH or Fv) and the Pearson correlation coefficient is indicated. (C)The Sc values for all VHH::HEWL and Fv::lysozyme complexes. The horizontalline through the bars represents the average Sc value for Fvs and VHHs.

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whose germ line genes for the H3 severely limit their length.Human and rabbit antibodies have, on average, longer H3 loopsand, therefore, might have a higher frequency of producingconvex paratopes. Indeed, structural data suggest that antiviralhuman antibodies (e.g., b12, 447–52D, 4E10, 2F5, 17b) alsotarget clefts or canyons by using long protruding H3 loops (28).

Our set of VHH::HEWL structures reveals that differentstructural solutions exist for binding the active site of HEWL.Despite their different epitope preference to that of murineanti-HEWL antibodies, most of the interface characteristics arestrikingly similar. In contrast, all VHHs, recognizing the HEWLactive site, display a convex paratope. It is the compact prolatenature of the VHH domain (due to absence of VL) and thecharacteristic H3 loop folding over the VHH framework regionthat generates a considerably convex antigen-combing site,possibly enhanced by a protruding loop structure as observed forcAb-Lys-3 (17) or by protruding side chains. It further providesan interaction surface as large as that of a combined VH-VL pairwhile leaving a significantly smaller footprint on the antigen.

Assuming that the pronounced convex paratope of a VHH isa key element for the preferential recognition of the HEWLactive site, other single domain antibody fragments shouldequivalently favor clefts on the surface of proteinaceous anti-gens. It was already reported that a considerable fraction of thedromedary HCAbs acts as potent �-amylase, carbonic anhydra-sen or �-lactamase inhibitors (19, 29). The crystal structure of aVHH against �-amylase confirmed that the convex paratope ofthe binder inserts into the enzyme’s active site (30). In addition,a llama VHH seemed to competitively inhibit potato starchbranching enzyme A (31). More strikingly, the single domain ofan anti-HEWL HCAb from nurse shark (i.e., NAR) recognizesalso the active site (32), where it overlaps with the epitope ofcAb-Lys-3. Here as well, the long H3 loop provides a largeconvex antigen interaction surface. Furthermore, a competitiveinhibitor for hepatitis C virus NS3 protease was derived from asynthetic library of ‘‘camelized’’ human VH domains (33). Theunique convex paratope of single domain antibody fragmentsdominated by the H3 loop residues to participate in the catalyticsite recognition offer a pharmacological benefit. It should befeasible to use the H3 antigen-binding loop as a lead to generatesmall peptide mimetics to develop agonists or antagonistsagainst medically important targets.

Of note, VHHs also form planar paratopes as exemplified bythe nonactive site binder D2-L24. Relatively small planarepitopes located outside clefts and enzyme active sites werealready documented for several high-affinity VHH-antigen as-sociations (34–37). Remarkably, the immunizations of drome-daries with bovine RNase A and human lysozyme failed so farto produce VHHs acting as competitive inhibitors (15, 19, 29).This absence might be explained, at least for these antigens, bya strong counterselection of self-epitopes and a redirection of theimmune response toward planar epitopes. Indeed, the sequencecomparison of bovine RNase A (38) and human lysozyme (39)with the equivalent dromedary enzymes (40, 41) revealed astrong conservation of the catalytic cleft residues.

MethodsIsolation of HEWL-Specific VHHs and Polyclonal Serum-VHH. Thedromedary D0 immunization in Morocco and isolation of thecAb-Lys-3 and cAb-Lys-2 have been described elsewhere (42).Dromedary D2 and D3 immunizations in Dubai and phage-displayed libraries of their VHHs (19) yielded six lysozyme-specific VHHs: D2-L19, D2-L24, D2-L29, D2-L27, D2-L31, andD3-L11. All VHH genes were recloned in a pHEN06 vector (29)for expression with a His-6 tail. The recombinant proteins wereproduced in Escherichia coli, and purified according to describedprotocols (29).

A polyclonal pool of monomeric VHHs was prepared from the

serum HCAbs. The HCAbs were first purified from dromedaryD2 (bleeding at day 54 in the immunization program; ref. 19) bydifferential adsorption on protein A�G chromatography (29).This IgG3 HCAb isotype was incubated with Staphylococcusaureus V8 endogluproteinase (Boehringer Mannheim) accord-ing to Lauwereys et al. (19) to prepare the monomeric polyclonalVHH.

Kinetic and Affinity Measurements of the VHH–HEWL Interaction. Thekinetic constants of the VHH–HEWL interactions were deter-mined by surface plasmon resonance (Biacore 3000). A CM5chip with �100 pg�mm2 (i.e., 100 resonance units) of immobi-lized HEWL was prepared according to published protocols(43). Association traces of five VHH concentrations (from 2000to 3 nM depending on the affinity) and a zero concentration(buffer) were recorded for 3 min in PBS, pH 7.4�0.005% Tween20�3 mM EDTA. Dissociation of the complexes was followed for10–20 min. A 30-s injection of 100 mM glycine�HCl (pH 1.5)regenerated the surface. Curves obtained after subtraction of thereference and buffer signals were fitted to a 1:1 Langmuirbinding model with BIAEVAL 3.1 (BIAcore). The KD value of theD2-L27 was determined by nonlinear regression of the equilib-rium binding signals versus the VHH concentration. The D2-L31–HEWL interaction was consistent with a model describinga conformational change upon binding.

Epitope Mapping and Interference of Binding by NAG3 and BiebrichScarlet. The epitopes of all VHHs were mapped on BIACORE3000 and IAsys. For this purpose, a CM5 chip with �300 pg�mm2

(300 resonance units) or an IAsys dextran curvette with �3,000pg�mm2 of immobilized HEWL was prepared according topublished protocols (18, 43). VHH–HEWL binding for eachVHH in the collection of HEWL specific binders was examinedin the presence of a different VHH member or in the presenceof the small lysozyme inhibitors GlcNAc(�1–4)GlcNAc(�1–4)GlcNAc (NAG3) and Biebrich Scarlet. This was accomplishedby using a coinjection procedure. Hereby, a HEWL saturatingconcentration of the VHH or small molecule was injected,followed by the injection of an identical sample supplementedwith a competing VHH. After regeneration of the surface, thecompeting VHH alone was applied. The binding levels of thecompeting VHH for both injections were then compared. Signaldifferences between both injections of at least 50% were con-sidered to originate from competition for overlapping epitopes.The concentrations of the inhibitors NAG3 (KD � 0.01 mM; ref.22) or Biebrich Scarlet (KD � 0.13 mM; ref. 22) were competitive(KD1�KD2 � C1�C2) with those of the VHH–HEWL association.

Structure Solution of the VHH–HEWL Complexes. X-ray diffractiondata of crystals of the D3-L11::HEWL, D2-L29::HEWL andD2-L27::HEWL complexes (crystallization conditions describedin Supporting Text, which is published as supporting informationon the PNAS web site) were obtained by using EuropeanMolecular Biology Laboratory (EMBL) beamlines at the Deut-sches Elektronen Synchrotron (DESY) synchrotron facility(Hamburg, Germany) or at the European Synchrotron Radia-tion Facility (ESRF, Grenoble, France). Data were processedwith DENZO and SCALEPACK (44). For the D3-L11::HEWL andD2-L24::HEWL data, AMORE (45) generated clear molecularreplacement solutions for HEWL and a CDR-deleted model ofcAb-RN05 [Protein Data Bank (PDB) ID code 1BZQ] (34) orcAb-Lys-3 (PDB ID code 1JTT) (16). For the data ofD2-L29::HEWL, PHASER (46) generated clear molecular re-placement solutions for HEWL and a CDR-deleted model ofcAb-An33 (PDB ID code 1YC7). The complete VHH sequenceswere built in the electron densities. Simulated annealing andrefinement protocols of CNS (47) refined the structures. Struc-ture determinations of cAb-Lys-3::HEWL (PDB ID code 1JTT),

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cAb-Lys-2::HEWL (PDB ID code 1RJC), and D2-L19::HEWL(PDB ID code 1RI8) were reported elsewhere (16, 43). Spacegroups, unit cells, data collection, and refinement statistics arepresented in Table 2.

Analysis of the VHH::HEWL and Fab::HEWL Interfaces. Several inter-face parameters were calculated for all VHH::HEWL complexes,and with identical settings for the six representativeFab�Fv::HEWL structures: F9.13.7::GEL (PDB ID code 1FBI);D11.15::JEL (PDB ID code 1JHL); D1.3::HEWL (PDB ID code1VFB); D44.1::HEWL (PDB ID code 1MLC); HyHEL-5::HEWL(PDB ID code 3HFL); and HyHEL-8::HEWL (PDB ID code1NDG). The change in solvent accessible surface area (�ASA) aswell as the polar fraction of the �ASA of the paratopes and epitopeswas calculated by the protein interaction server (2). The server alsoprovided the epitope�paratope planarity index, defined as the rootmean square deviation of the epitope�paratope atoms from thebest-fit plane through their coordinates. CONTACT from the CCP4software (48), tabulated the antibody–lysozyme atom pairs withina contact distance of 4 Å, of which (oppositely) charged pairs were

considered to form salt bridges. HBPLUS (49) identified potentialantibody–lysozyme hydrogen bonds and bridging solvent molecules.The paratope–epitope complementarities were estimated by cal-culating the Sc values (50) for solvent-excluded antibody::lysozymecomplexes. The Mann–Whitney U test, a nonparametric alternativefor the parametric t test, implemented in SPSS FOR WINDOWS(release 12.0, 2003, SPSS Inc., Chicago), tested the significance ofobserved differences in interfaces parameters betweenVHH::HEWL and Fv::lysozyme interfaces. Figures were preparedwith PYMOL (www.pymol.org). Amino acid sequences were num-bered according to Kabat et al. (51).

We acknowledge D. Maes for running the statistical significance tests.This research was sponsored by Fonds voor Wetenschappelijk Onder-zoek–Vlaanderen, Vlaams Interuniversitair Instituut voor Biotechnolo-gie, and Geconcerteerde Onderzoeks Actie (Research Council, VrijeUniversiteit Brussel). We acknowledge the use of the synchrotron beamtime at European Molecular Biology Laboratory (EMBL) beam lines atthe DORIS storage ring (Hamburg, Germany) and EMBL beam lines atESRF (Grenoble, France).

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